July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Transforming growth factor beta 3 of human corneal stromal stem cells plays a pivotal role in suppression of mouse corneal scar formation
Author Affiliations & Notes
  • Lin Weng
    Department of ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Martha L Funderburgh
    Department of ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Irona Khandaker
    Department of ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Moira Geary
    Department of ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Katherine Davoli
    Department of ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • James Funderburgh
    Department of ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States
  • Footnotes
    Commercial Relationships   Lin Weng, None; Martha Funderburgh, None; Irona Khandaker, None; Moira Geary, None; Katherine Davoli, None; James Funderburgh, None
  • Footnotes
    Support   NIH Grant EY016415, P30-EY008098, Research to Prevent Blindness, Eye and Ear Foundation of Pittsburgh
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2289. doi:
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      Lin Weng, Martha L Funderburgh, Irona Khandaker, Moira Geary, Katherine Davoli, James Funderburgh; Transforming growth factor beta 3 of human corneal stromal stem cells plays a pivotal role in suppression of mouse corneal scar formation. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2289.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Therapy using human corneal stromal stem cells (CSSC) can significantly suppress scar formation in healing corneal wounds. However, the molecular mechanism is not fully understood. Transforming growth factor beta 3 (TGFb3) is reported to contribute to scarless healing of fetal wounds. In this study we probed the potential that TGFb3 of CSSC plays a role in the suppression of scarring by these cells.

Methods : CSSC were co-cultured in transwells with macrophage cell line RAW 264.7 (RAW) in 10% FBS DMEM/F12 containing IFN-gamma and LPS to induce the M1phenotype or IL-4 to induce M2. Expression of human TGFb1 and TGFb3 by CSSC and mouse iNOS and ARG-1by RAW cells was analyzed using qPCR. TGFb3 expression was knocked down in CSSC by siRNA transfection using Viromere Blue. Expression levels of TGFb1 and TGFb3 in healing mouse cornea with or without CSSC was analyzed using Nanostring assay and Taqman qPCR. Corneal scarring was evaluated using Optical coherence tomography (OCT) image analysis. Fibrotic gene expression in wounded corneas analyzed using qPCR. TGFb3 protein deposition was visualized by immunostaining whole-mounts of wounded corneas. Significance of quantitative values was determined by one-way ANOVA.

Results : In co-culture with RAW macrophages, a 30-fold increase of TGFb3 gene expression by CSSC was observed after 48 hours under M1 conditions but not in M2-inducing medium. CSSC TGFb1 expression remained steady in both co-culture conditions. RNA for human TGFb3 was detected in wounded corneas treated with CSSC, but Nanostring analysis showed no difference in expression of mouse TGFb1 or TGFb3, 1-15 days after wounding comparing wounds with and without CSSC. Corneal wounds treated with CSSC exhibited immunostaining for TGFb3 localized near engrafted CSSC cells. OCT analysis showed wounded mouse cornea treated withCSSC with TGFb3 knockdown to have significantly greater scarring (n=12, p< 0.003) and increased expression fibrotic genes (Acta2, Col3a1, Fn1) compared to wounds treated with CSSC transfected with control siRNA (n=3, p<0.001).

Conclusions : Our results support the hypothesis that TGFb3 of CSSC is induced in a wound healing environment, possibly resulting from interactions with infiltrating immune cells. Secretion of TGFß3 appears toplay an role in suppression of corneal scar formation.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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